Rosmarinus officinalis extract suppresses Propionibacterium acnes-induced inflammatory responses

Abstract

Propionibacterium acnes is a key pathogen involved in the progression of acne inflammation. The development of a new agent possessing antimicrobial and anti-inflammatory activity against P. acnes is therefore of interest. In this study, we investigated the inhibitory effect of rosemary (Rosmarinus officinalis) extract on P. acnes-induced inflammation in vitro and in vivo. The results showed that ethanolic rosemary extract (ERE) significantly suppressed the secretion and mRNA expression of proinflammatory cytokines, including interleukin (IL)-8, IL-1β, and tumor necrosis factor-α in P. acnes-stimulated monocytic THP-1 cells. In an in vivo mouse model, concomitant intradermal injection of ERE attenuated the P. acnes-induced ear swelling and granulomatous inflammation. Since ERE suppressed the P. acnes-induced nuclear factor kappa-B (NF-κB) activation and mRNA expression of Toll-like receptor (TLR) 2, the suppressive effect of ERE might be due, at least partially, to diminished NF-κB activation and TLR2-mediated signaling pathways. Furthermore, three major constituents of ERE, carnosol, carnosic acid, and rosmarinic acid, exerted different immumodulatory activities in vitro. In brief, rosmarinic acid significantly suppressed IL-8 production, while the other two compounds inhibited IL-1β production. Further study is needed to explore the role of bioactive compounds of rosemary in mitigation of P. acnes-induced inflammation.

Figures

FIG. 1.

Effects of ethanolic rosemary extract (ERE) on production (A) and mRNA expression (B) of pro-inflammatory cytokines in Propionibacterium acnes–stimulated monocytic THP-1 cells. (A) Cells were co-incubated with dimethyl sulfoxide (DMSO; as vehicle) or the indicated concentration of ERE and viable P. acnes for 24 h. A control experiment without P. acnes treatment was done in parallel. The culture supernatants were subsequently isolated and analyzed for cytokine production. (B) The expression level of mRNA was determined using quantitative real-time polymerase chain reaction. The expression of cytokine mRNA was normalized to GAPDH mRNA and expressed as multiples of change with untreated THP-1 cells as the control. Each column shows the mean±standard deviation (SD) of three independent experiments with triplicate wells in each. *Significant difference from vehicle (P. acnes alone) at P<.05 by Student's t-test.

FIG. 2.

Inhibitory effect of ERE (1 mg) on P. acnes–induced inflammation. (A) Ear biopsies of ICR mice which were intradermally injected with PBS vehicle (i), P. acnes(ii), ERE (iii), or ERE+P. acnes(iv) were observed after hematoxylin and eosin staining. Scale bars represent 200 μm. The inhibitory effects of ERE on P. acnes–induced ear edema in mice were evaluated by measuring the ear thickness (B) and ear biopsy weight (C). *Significant difference from vehicle (P. acnes alone) at P<.05 by Student's t-test. Color images available online at www.liebertpub.com/jmf

FIG. 3.

Effect of ERE on P. acnes–induced nuclear factor-κB (NF-κB) activation in THP-1 cells. THP-1 cells were incubated for 8 h (A) and 16 h (B) without P. acnes (control), with P. acnes alone (DMSO vehicle), and with P. acnes in the presence of the rosemary extract. Data are presented as the mean±SD. *Significant difference from vehicle (P. acnes alone) at P<.05 by Student's t-test.

FIG. 4.

Effect of ERE on toll-like receptor 2 (TLR2) mRNA expression. (A) ERE did not affect the constitutive TLR2 expression of THP-1 cells in the absence of P. acnes after 24h-incubation. (B)P. acnes induced TLR2 mRNA overexpression in THP-1 cells for 8 h and 16 h incubation. *P<.05 compared to time zero (baseline). (C) THP-1 cells were incubated for 8 h (i) and 16 h (ii) untreated with P. acnes (control), and treated with P. acnes alone (DMSO vehicle) and with P. acnes in the presence of the rosemary extract. The TLR2 mRNA expression was normalized to GAPDH mRNA and expressed as multiples of change with untreated THP-1 cells as the control. Data are presented as the mean±SD. *Significant difference from vehicle (P. acnes alone) at P<.05 by Student's t-test.

FIG. 5.

HPLC profiles of standard phenolic compounds (A) and phenolic compounds in ERE (B). Detection was at 284 nm. Peaks: 1, rosmarinic acid; 2, carnosol; 3, carnosic acid; d, unidentified.

FIG. 6.

Effects of carnosol, carnosic acid, and rosmarinic acid on proinflammatory cytokine production in P. acnes–stimulated monocytic THP-1 cells. Cells were coincubated with DMSO (as vehicle) or the indicated concentration of samples and viable P. acnes for 24 h. A control experiment without P. acnes treatment was conducted in parallel. The culture supernatants were subsequently isolated and analyzed for cytokine production. Data are presented as the mean±SD. * Significant difference from vehicle (P. acnes alone) at P<.05 by Student's t-test.